1. Field of the Invention
The present invention relates to disulfide-stabilized (ds) recombinant polypeptide molecules, such as the variable region of an antibody molecule, which have the binding ability and specificity for another peptide. Methods of producing these molecules and nucleic acid sequences encoding these molecules are also described.
2. In the Background
Antibodies are molecules that recognize and bind to a specific cognate antigen. Numerous applications of hybridoma-produced monoclonal antibodies for use in clinical diagnosis, treatment, and basis scientific research have been described. Clinical treatments of cancer, viral and microbial infections, B cell immunodeficiencies, and other diseases and disorders of the immune system using monoclonal antibodies appear promising. Fv fragments of immunoglobulins are considered the smallest functional component of antibodies required for high affinity binding of antigen. Their small size makes them potentially more useful than whole antibodies for clinical applications like imaging tumors and directing recombinant immunotoxins to tumors since size strongly influences tumor and tissue penetration.
Fv fragments are heterodimers of the variable heavy chain domain (V.sub.H) and the variable light chain domain (V.sub.L). The heterodimers of heavy and light chain domains that occur in whole IgG, for example, are connected by a disulfide bond. The Fv fragments are not and therefore Fvs alone are unstable. Glockshuber et al., Biochemistry 29:1362-1367 (1990). Recombinant Fvs which have V.sub.H and V.sub.L are connected by a peptide linker are typically stable, see, for example, Huston et al., Proc. Natl. Acad, Sci. USA 85:5879-5883 (1988) and Bird et al., Science 242:423-426 (1988). These are single chain Fvs which have been found to retain specificity and affinity and have been shown to be useful for imaging tumors and to make recombinant immunotoxins, for tumor therapy for example. However, researchers have bound that some of the single chain Fvs have a reduced affinity for antigen and the peptide linker can interfere with binding.
Another approach to stabilize the Fvs was attempted by Glockshuber et al., supra. Disulfide bonds were placed in the complementarity determining regions (CDR) of an antibody whose structure was known in a manner that had limited or no effect on ligand binding. This approach is problematic for stabilizing other Fvs with unknown structures because the structure of each CDR region changes from one antibody to the next and because disulfide bonds that bridge CDRs will likely interfere with antigen binding. Thus, it would be desirable to have alternative means to stabilize the Fv portions of an antibody of interest which would allow the affinity for the target antigen to be maintained.